Separation processes are used widely in biological science to isolate one component from complex mixtures using a single or combination of unique characteristics of the desired entity. These characteristics can be size, shape, charge, hydrophobicity, solubility, density etc. all coming into the categorisation of chromatography. Usually these characteristics are not totally unique and so a series of different sequential separations must usually be taken to refine the purification. Often the choice of which steps to use is empirically determined and devising a new purification route is very laborious, each choice having many variants within it. The labile nature of many biological substances also makes this a difficult procedure, the desired substance sometimes decaying as fast as it can be purified.
A variant of these techniques is affinity chromatography. In this a more specific characteristic of the desired item is utilised in the separation strategy. Usually this involves a specific binding capability which is instrumental in effecting the separation. Biological systems employ specific binding very regularly as part of their natural functioning, such as in antibody/antigen interactions in disease resistance, or receptor/ligand interactions for cell signalling. This can be harnessed in a separation technique to obtain 100% separation in one step and is therefore a particularly powerful method. With such a powerful separation method it is important when performing these activities that all contaminating unbound materials are removed with high efficiency. Preferably there should be 100% efficiency both of contaminant removal and of capture of desired substance in the first pass. This requires maximal interaction between the desired substance or entity with its binding partner as well as very efficient washing.
A method is therefore needed where sufficient capture molecules to capture all the target molecules are held on a solid phase. This is done in such a way that the target molecule or entity has easy access to the binding site but also so that non-binding moieties can be washed off vigorously without trapping or spuriously interacting.
There are many formats for performing affinity chromatography procedures. All of them share the general feature of having one side of the binding pair immobilised to a solid phase. This most commonly consists of a bead material sometimes packed into columns. The liquid containing the desired entity can then flow over, around and in some cases inside the beads coming into contact with the capture entity and contaminants can then be washed by allowing washing solutions to flow over the bead surfaces. The stringency of the washing procedure can be influenced by the nature of the washing solution such as by temperature, ionic strength, pH, solvent mix etc.
Beads are in many cases a preferred option as this format maximises the surface on which the capture entity is immobilised.
In general the major requirement is for an insoluble material to which the capture entity can be attached such that a fluid containing the target be passed over the solid phase to allow maximum contact between the compartments. Also the solid phase normally requires a maximal surface area to allow sufficient capture entity to be available. In most cases the purpose of the procedure is for the removal of the target from a complex mixture in pure form and then elution of the target from the capture entity without the capture entity also being removed. For this to be possible conditions have to be found where the binding forces can be overcome without damage to either binding partners.
In recent times, especially in molecular biology, techniques have arisen which are manyfold more sensitive than before and so do not need such large amounts of sample as starting material. Also more and more of subsequent treatments are able to be done while still attached to the solid phase. This is the basis of the burgeoning fields of downstream processing and biotransformations where chemical modifications are made often enzymically by materials held on a solid phase.
There have appeared in the last few years several new formats for affinity chromatography based on filtration membranes especially for antibody purification. Most commonly these involve the use of a filtration cartridge format, familiar to those working in biological fields. This consists of either a disposable or reusable cassette within which is mounted a disc of filtration membrane.
The membrane is supported on both sides by plastic meshes within the cassette and leading out from the upper and lower surfaces of the cassette is a nozzle designed to be attached to a syringe and an outlet designed to be directed into the collection vessel. These cassettes sometimes include in the design, channels of liquid flow to maximise the interaction of the fluid across the membrane (U.S. Pat. No. 4,690,757).
Later developments have lead to new versions with either capture moieties already permanently attached or in a chemically activated form for custom derivatisation. The discs are usually about 5 cm in diameter and are claimed to have as high a binding capacity as a column. This is consistent with their use with a syringe for the application of large samples of between 1 and 50 mls of solution at a time.
As mentioned earlier the trend now is towards smaller samples, more sensitive detection and amplification techniques especially in molecular biology. In biology large samples are difficult to obtain and involve significant derangement to the biological entity which is the source of the sample. This is especially true if repetitive samples need to be taken to follow a trend or reaction. Large samples are also slower to process and involve exposing fragile biological entities to inhospitable environments during the process.
As mentioned earlier existing affinity processes involve removal of the target from the capture entity as the final step. This requires empirical discovery of conditions which will perturb the binding without damaging the desired purified moiety. This can be extremely difficult for example with antibodies where strong binding is often particularly desirable. Obviously the stronger the binding the more denaturing the eluting solution. Sometimes numerous combinations of elutant have to be tried to find a good set of conditions. Sometimes however no combination achieves the right effect.
As well as molecular purifications cells are also used in affinity processes. This can take a variety of forms based on different characteristics of the cells. Usually but not always the cells must be recovered intact from the process for further analysis. Frequently the separation is based on presence of cell surface molecules for which antibodies can be obtained. This can also be combined with size and density measurements. Methods for affinity purification of cells include "Fluorescence Activated Flow Cytometry" which can combine size with the existence of one or more cell surface markers. The problems of cell purification are severe due to their fragility and if antibody selection has been used the selected cells have to be used with the label still attached.
Many areas of science use natural affinities for binding. In genetics complementarity of nucleic acids is commonly utilised as the basis of a method of analysis. For example mRNA is isolated by virtue of the fact that it always has a tail of adenine nucleotides at the end which can be bound to a row of thymidine nucleotides.
Specific gene nucleotide sequences can be captured by the complementary nucleotide sequence. These hybrids can usually be removed very easily by reducing the ionic strength of the elutant allowing the natural charge-driven repulsion between DNA strands to take effect.
As mentioned earlier, sometimes elution conditions cannot be found to remove without damage. This can be turned into an advantage however if the capture is done in the situation where it can act as the linker immobilising the desired activity in place so that subsequent steps can be performed in situ. For example this could be an enzyme reaction in the new science of biotransformations which uses immobilised enzymes for chemical synthesis.
If it is essential to remove the target material then a final resort is to use a membrane material which is itself soluble in a solvent not damaging to the target. This case however does release the capturing material also.
Frequently the coupling of the material to its solid phase would be by covalent linker to avoid any problems of the leaching out of the capture moiety leading to contamination of the process.
If removal of the target moiety is difficult or unnecessary, analytical work can be done in situ. Many biological analyses can be performed on membranes. For example one item is immobilised on the membrane and used to capture the other. Using further labelled binding moieties the presence of an entity can be revealed using fluorescent markers or colourimetric enzyme markers or radiolabels. These can be visualised by eye, by machine or by microscopic analysis or counted by appropriate instrumentation. The requirement for being on a membrane is to allow efficient washing as well as to provide for information of localisation.
Other processes are now carried out on membranes such as enzyme reactions, gene amplification reactions, chemical syntheses etc.
Often the results of a separation especially if it involves cells must be verified by direct observation. This is used to tell whether the cells are still in good condition, whether the separation looks "clean" etc. Sometimes further specific tests must be done on the separated cells to verify their identity by a different route such as specific staining or enzyme activity. If a cell sorter has been used then the produced cells can be examined. If a column has been used they must be eluted to be observed. If however a membrane has been used they can be visualised directly as most membranes are semi-transparent or translucent.
Most capture-based techniques still utilise the column format. This requires the sample to be slowly trickled over the matrix and then washing to be done by trickling over the matrix a succession of washing steps. To improve the washing and the elution, gradients of solvents are often used with varying pH, ionic strength and hydrophobicity.
Also several changes of small volumes of wash solution are far more efficient than one large one which is difficult to implement in a column situation without extending the time further.
These processes can take a long time especially if the binding affinity is weak and sometimes requires the circulation of the sample solution over and over the capture surface to maximise contact. During this long time many constituents will deteriorate and possible denature and consequently many of these processes are now carried out in cooled rooms. These are very unpleasant environments to work in and only partially solve the problem.
One solution to this has been the development of HPLC techniques which among their other characteristics are faster, as the liquids are transferred under pressure. These systems are expensive however and subject the substances to high pressures as well as temperature.
Membrane capture processes are usually faster and therefore better for labile materials but they suffer from problems of dead space which means that the smallest samples cannot easily be used and that the material eluted is lower in concentration.
As existing cartridges are contained, it is not easy to see when they are full of liquid and this can result in air being drawn through and partial drying out of the membrane in an attempt to reduce the minimum volume. Also, because the membranes are contained and supported it is not easy to remove the membrane for visualisation either by light electron microscopy. Similarly they cannot easily be used for subsequent reactions. Some of the cartridges can be disassembled and hence the membrane removed. The true purpose of this is re-use of the cartridge however and usually results in some damage to the membrane.
In some samples the desired constituent is present in minute amounts or numbers. This results in large volumes being drawn over the capture moieties. In addition to the time involved this has the additional disadvantage that the process of liquid flowing in some cases is sufficient to cause the removal of hitherto bound components. The severity of this depends on the nature and strength of the binding, but as biologically significant affinities are often subtle there are many cases where this method of purification cannot be achieved for these reasons.
Another route to solving these problems is to increase the amount of available capture partner by increasing the amount of solid phase. This results in slower flow rates, longer reaction time and greater dilution of the desired material.
Samples for these types of purifications are often clinical and potentially infective and the washing and especially eluent chemicals are also frequently of a hazardous nature. They may be organic solvents, acids, ion-pairing molecules, chelators, detergents etc. Traditional processes using columns offer the potential of injury to the operators as they are exposed to the whole system which often involves the use of significant amounts of the hazardous material. Membrane cartridge devices are better but still rely on squirting out liquids with possibilities for spillages and aerosols.
Many targets once purified will be used in further analysis such as electrophoresis or reactivity for example. For most of these subsequent reactions the target, which is usually in limiting amounts, is preferably at a high concentration. For column and membrane affinity systems, elution will result in the sample being collected at less than maximal concentration. This frequently means that concentration has to be performed before further work can be done. The concentration of a highly purified substance at low starting concentration is very inefficient often leading to losses in excess of 50%.
These small amounts of dilute material also suffer from the disadvantage of being easily denatured on storage and usually have to be mixed in with other molecules such as bovine serum albumin to increase their stability. This defeats some of the object of purifying them in the first place. They are also very liable to adsorb to the surfaces of their storage vessels which sometimes necessitates pre-treatment with toxic silicon compounds (silanes) as a prevention.
As mentioned earlier most chromatographic procedures are reached empirically and frequently involve multiple stages. Strategies are designed by the individual testing of single steps under a variety of conditions on a small scale to optimise both the type and order of steps sometimes for subsequent large scale operations.
This usually means that large numbers of a variety of small columns have to be made, equilibrated, and run both singly and in combination and the elution analysed and monitored. Constituents often need to be radio-labelled to be able to detect them in such processes and this results in a considerable amount of radio-active waste. There are automated systems to perform these reactions but they are very expensive, complex to run and the produced materials still need to be analysed.
The concept of having a separation on the end of a tip has been utilised before in patent specification No. WO8809201. In this case however the tip contains column material between two frits and is therefore a miniature column. Usage of the column is by solution flowing by gravity as commonly employed for column processes.